Methylation-associated inactivation of RASSF1A has frequently been observed in several human malignancies including sporadic colorectal and gastric cancer. However, nothing is known about the RASSF1A methylation status in the setting of MMR-deficient gastrointestinal tumours. In this study, we analysed systematically alterations in KRAS, BRAF and RASSF1A, in order to define the frequency and the pattern of these genetic/epigenetic alterations in three distinct subsets of MSI gastrointestinal tumours. Further, an association study was performed between RASSF1A methylation and the clinicopathological parameters in order to determine the profile of tumours harbouring this epigenetic event. A total of 56 MSI sporadic gastrointestinal tumours (31 colorectal and 25 gastric) and 20 MSI HNPCC analysed for KRAS/BRAF were analysed for RASSF1A promoter hypermethylation by MSP and DNA sequencing. No significant differences were found between the frequency of RASSF1A methylation in sporadic MSI colorectal and gastric carcinomas and HNPCC carcinomas (P=0.31). Methylation of RASSF1A was present in 16 of 31 (52%) sporadic MSI colorectal and 11 of 25 (44%) MSI gastric carcinomas, and in six of 20 (30%) HNPCC carcinomas. Nearly 36% of MSI sporadic colorectal carcinomas (CRCs) had RASSF1A methylation and activating mutations at KRAS and/or BRAF. In contrast, only 10 and 8% of HNPCC and sporadic gastric carcinomas, respectively, had concomitant KRAS mutations and RASSF1A methylation. The MSI sporadic gastric and CRCs with RASSF1A methylation were preferentially poorly differentiated (P=0.03, 0.05, respectively). We show that the profile of alterations RASSF1A, KRAS/BRAF is different among the three groups of MSI gastrointestinal tumours. Further, we demonstrate that MSI sporadic CRCs accumulated significantly more epigenetic/genetic alterations in RASSF1A, KRAS/BRAF than MSI sporadic gastric or HNPCC carcinomas (P=0.016). These results are likely to have therapeutic implications in the near future, due to the possibilities of using specific kinase inhibitors alone or in association with demethylating agents in MSI tumour types harbouring KRAS or BRAF mutations and RASSF1A methylation.